Aβ immunohistochemistry

Immunostaining was performed as described previously (22, 24). Briefly, brains were coronally sectioned through the cortex and hippocampus, and staining was performed on 40-μm free-floating sections using a peroxidase-based immunostaining protocol (VECTASTAIN Elite ABC HRP Kit). Sections were incubated overnight in primary antibody for Aβ (1:200) at 4°C, washed, incubated in biotinylated antibody (biotinylated horse anti-rabbit, 1:400) for 90 min at 4°C, then incubated in an avidin biotin enzyme reagent for 90 min at 4°C, and visualized using a chromogen. Sections were mounted on slides and visualized with a Zeiss AxioObserver epifluorescence microscope with a Zeiss 20× lens, using representative 900-μm² areas of cortex and hippocampus. Experimenters were blinded to drugging and analysis. Six to eight sections per mouse were analyzed, and for each section, five ROIs were analyzed in the cortex and two ROIs in the hippocampus using the cell counter tool in ImageJ (54). This number of ROIs prevents the selection of only densely stained regions.